Alkaline Protease Production from Bacillus gibsonii 6BS15-4 Using Dairy Effluent and Its Characterization as a Laundry Detergent Additive

Protease is a widely used enzyme particularly in the detergent industry. In this research, we aimed to isolate alkaline protease-producing bacteria for characterization as a laundry detergent additive. The screening of alkaline protease production was investigated on basal medium agar plus 1% skim milk at pH 11, with incubation at 30°C. The highest alkaline protease-producing bacterium was 6BS15-4 strain, identified as Bacillus gibsonii by 16S rRNA gene sequencing. While the optimum pH was 12.0, the strain was stable at pH range 7.0-12.0 when incubated at 45°C for 60 min. The alkaline protease produced by B. gibsonii 6BS15-4 using dairy effluent was characterized. The optimum temperature was 60°C and the enzyme was stable at 55°C when incubated at pH 11.0 for 60 min. Metal ions K+, Mg2+, Cu2+, Na+, and Zn2+ exhibited a slightly stimulatory effect on enzyme activity. The enzyme retained over 80% of its activity in the presence of Ca2+, Ba2+, and Mn2+. Thiol reagent and ethylenediaminetetraacetic acid did not inhibit the enzyme activity, whereas phenylmethylsulfonyl fluoride significantly inhibited the protease activity. The alkaline protease from B. gibsonii 6BS15-4 demonstrated efficiency in blood stain removal and could therefore be used as a detergent additive, with potential for various other industrial applications.

microbial growth on inexpensive media, easily produced on a large scale, economically feasible, with a high production yield whilst exhibiting high activity and stability at wide ranges of pHs and temperatures [5].
Alkaline proteases are a type of serine proteases, containing a serine and histidine as essential catalytic amino acid residues at their active sites, and catalyze hydrolytic reactions affecting amides and esters as well as peptides. Many alkaline proteases with high activity and stability in high alkaline range (pH 9-12) are interesting for numerous biotechnological and bioengineering applications [3]. They are a large group of enzymes, widely used in the detergent, food, pharmaceutical, and leather industries [6,7]. In particular, alkaline proteases are an essential ingredient for the detergent industry. They are used as a supplementary ingredient in different brands of commercial detergents to increase cleaning efficiency by eliminating proteins such as milk, blood, and food stains. The action of alkaline proteases in a detergent depends on many factors such as pH, temperature, detergent composition, and the type of washing machine used. The key to utilizing alkaline protease as a complement in detergent commodities is the stability of the enzyme in the detergent. Therefore, we sought to identify a new type of alkaline protease with good, functional and stable properties that make it suitable as an additive in detergents.
Even though numerous microorganisms are identified as producing alkaline proteases, Bacillus spp. are the preferred main sources for commercial and industrial enzymes owing to their exceptional ability to secrete large amounts of highly active enzymes, whilst some of these genera are generally regarded as safe. The genus Bacillus, belonging to the family Bacillaceae, is gram-positive, rod-shaped, and endospore-forming [21]. In addition, Bacillus plays a particularly role in industry; it is the best producer of alkaline protease with pH activity values above 7.0, while approximately half of all commercial protease production is obtained from Bacillus spp. [22]. Therefore, the isolation and screening of new, alkaline protease-producing Bacillus strains from natural habitats, could pave the way for new opportunities and developments. The isolated strains could be used for observing and producing novel alkaline protease in various industries and in highly alkaline conditions. This research aimed to screen, isolate, identify, and characterize the excellent, alkaline protease-producing bacteria collected from the Resources Protection Area of Khok Phutaka, Khon Kaen Province, under the Plant Genetics Conservation Project under the Royal Initiation of her Royal Highness Princess Maha Chakri Sirindhorn. In addition, dairy effluent was used as a raw material to produce alkaline protease from selected bacteria because it is cheap and rich in nutrients. Finally, the efficiency of the crude enzyme as a detergent additive was also evaluated.

Isolation of Alkaline Protease-Producing Bacteria
Soil samples collected from the Resources Protection Area in Khok Phutaka, Khon Kaen Province under the Plant Genetics Conservation Project under the Royal Initiation of her Royal Highness Princess Maha Chakri Sirindhorn were diluted and homogenized in 0.9% sterile saline solution. The suspension was streaked on basal medium plus 1% skim milk agar plate (BMSM: 1.0% glucose, 1% skim milk, 0.5% yeast extract, 0.1% K 2 HPO 4 , 0.02% MgSO 4 .7H 2 O, 1.0% Na 2 CO 3 , agar 1.5%, pH 11) [23] and incubated at 30°C for 72 h. The alkaline protease production bacteria were observed for protein hydrolysis by formation of clear zones around the colonies. The diameters of the bacterial colonies (CO) and the clear zones around the bacterial colonies (CZ) were measured. The difference between CZ and CO (CZ -CO) and the ratio between CZ and CO (CZ / CO) were compared. The selected strains with maximum high values of both calculations were further analyzed for alkaline protease activities.

Alkaline Protease Production and Enzyme Assay
The selected strains were cultured in BMSM, pH 11, and incubated at 30°C with shaking at 120 rpm for 0-192 h. Samples were collected every 24 h to analyze cellular growth and alkaline protease activity. The cell culture was centrifuged (10,000 ×g, 10 min) and the supernatant was recovered as crude protease for analysis. The cell sediment was determined for cellular growth by viable plate count.
Alkaline protease assay was measured by the modified method using casein as a substrate [24]. The mixture, a diluted enzyme 1 ml and 1 ml of 1% casein (in glycine-NaOH buffer, pH 11.0) was incubated at 45°C for 15 min followed by the addition of 2 ml of 10% trichloroacetic acid (TCA). It was then left at 4°C for 1 h to allow complete or almost complete precipitation of protein. The control sample was also prepared by adding TCA before the enzyme addition. The reaction mixture was centrifuged, and the supernatant was measured for tyrosine concentration [25]. One unit of enzyme alkaline protease was defined as the amount of protease that released 1 μg/ml of tyrosine from casein per minute under experiment conditions.

Identification of Alkaline Protease-Producing Bacteria
Molecular identification of the selected strain was achieved by sequence analysis of 16S rRNA gene. The 16S rRNA gene was amplified using forward primer 785F (5′-GGA TTA GAT ACC CTG GTA-3′) and reverse primer 907R (5′-CCG TCA ATT CMT TTR AGT TT) 3′). The PCR products were purified and sequenced by Macrogen Inc., Korea. The sequence was submitted, and the accession number was provided by GenBank.

Preparation of Inoculum and Alkaline Protease Production on Dairy Effluent
Alkaline protease production was carried out in two steps. First, the selected strain was cultured in BMSM, pH 11, and incubated at 30°C with shaking at 120 ×g for 24 h. Second, the seed culture was inoculated (1%v/v) into 100 ml of dairy effluent supplement with basal medium (1.0% glucose, 0.5% yeast extract, 0.1% K 2 HPO 4 , 0.02% MgSO 4 .7H 2 O, 1.0% Na 2 CO 3 ) plus 0, 0.25, or 0.5% skim milk, and the pH was adjusted to 11 before incubation at 30°C with shaking at 120 rpm for 168 h. The enzyme was collected for alkaline protease characterization and compared with basal medium plus 1% skim milk (conventional media) cultivation.

Effect of pH and Temperature on Enzyme Activity and Stability
Alkaline protease activity was measured at a pH range of 3 to 13 at 45°C for 15 min using casein as a substrate. The pH stability was determined by preincubation in various buffer solutions with different pH values for 60 min at 45°C. The enzymes were withdrawn, and remaining enzymatic activities were determined at pH 11 and 45°C. The four buffers system used in this study consisted of 0.05 M acetate buffer (pH 3-6), 0.05 M phosphate buffer (pH 6-8), 0.05 M glycine-NaOH buffer (pH 8-11), and 0.05 M hydroxide-chloride buffer (pH [11][12][13].
The optimum temperature of enzyme activity was assessed at between 30 and 80°C using casein as a substrate in 0.05 M glycine-NaOH buffer, pH 11. Temperature stability was investigated by incubating the enzyme in 0.05 M glycine-NaOH buffer, pH 11.0, for 60 min over a temperature range of 30 to 80°C. The enzymes were withdrawn, and remaining enzymatic activities were determined at pH 11 and 45°C.

Washing Performance Test with Alkaline Protease
The efficiency of alkaline protease (0.316 units) as a detergent additive was studied on white cotton cloth pieces (5 × 5 cm) stained with bovine blood. Fresh bovine blood (100 μl) was spread on the surface of each piece of white cotton cloth, and the pieces were left in the oven (60°C) for 4 h to dry and fix the stain. The following set experiments were prepared and tested with (a) 200 μl distilled water, (b) 200 μl glycine-NaOH buffer, pH 11.0, (c) 100 μl crude enzyme plus 100 μl detergent, (d) 100 μl detergent solution100 μl glycine-NaOH buffer, pH 11.0, and (e)100 μl crude enzyme plus 100 μl glycine-NaOH buffer, pH 11.0. Each condition was dropped on the bovine blood-stained cotton pieces and incubated at 45°C for 60 min. After incubation, each piece of cloth was washed with tap water for 2 min and air-dried for 4 h. Untreated bovine blood-stained cotton pieces with enzyme were taken as control. The blood stain removal was determined by visualization.

Hydrolysis of Protein Substrates
The ability of alkaline protease enzyme to hydrolyze blood, eggs, chicken feathers and milk were investigated. The samples were mixed with alkaline protease enzyme (158.44 units). The mixtures were incubated at 30°C for 3 days and samples were taken every day to determine the protein hydrolysis ability. The amino acid from the protein solution for each hydrolysis reaction was determined by using the Lowry method.

Isolation of Alkaline Protease-Producing Bacteria
Few cultures appeared owing to the high alkaline condition of the isolation media. The alkaline protease production bacteria were observed for protein hydrolysis by formation of clear zones around the colonies. Photographs of protein hydrolyzed zones are presented in Fig. 1. The 169 isolates showing a clear zone of proteolysis were point inoculated on BMSM agar for calculating the difference between CZ and CO (CZ -CO) and the ratio between CZ and CO (CZ / CO). Among those isolates, the 13 isolates which obtained high values of both (CZ -CO) and (CZ / CO) ratio were selected for further alkaline protease determination. Bacterial isolate 6BS15-4 showed the highest difference between CZ (27.5 mm) and CO (6.3 mm) and the greatest ratio between CZ and CO, at 21.2 mm and 4.37, respectively.
The 13 isolates were further screened quantitatively for the production of alkaline protease in BMSM broth with the shake flask. The highest alkaline protease-producing bacterium was 6BS15-4 strain (316.88 units/ml) within 168 h of cultivation. Alkaline protease production started after a 24-h lag phase, then increased exponentially along with alkaline protease production from 24-48 h. From stationary phase to dead phase, cellular growth and enzyme production were exhibited in the range 48-192 h of cultivation. Time course of the cellular growth and alkaline protease production of 6BS15-4 strain are shown in Fig. 2. In addition, 6BS15-4 strain was identified as Bacillus gibsonii by 16S rRNA gene sequencing. The results revealed that strain 6BS15-4 exhibited a high level of 16S rRNA gene similarity (99%) with B. gibsonii NR_026143.1.

Alkaline Protease Production on Dairy Effluent
Dairy effluent supplement was collected from a dairy cow farm. The total protein and reducing sugar were observed at 766.60 mg/l and 381.30 mg/l, respectively. Relative activity of alkaline protease production from B. gibsonii 6BS15-4 when cultured in various culture media compared with conventional medium are shown in Table 1.
Alkaline protease activity production from B. gibsonii 6BS15-4 using dairy effluent supplement with basal medium obtained 72.95% relative activity of alkaline protease activity when compared with alkaline protein cultivation from conventional media. Moreover, enzyme production had few differences when cultured in dairy effluent supplement with basal medium plus 0, 0.25, or 0.5% skim milk. Therefore, dairy effluent supplement with basal medium could be used for enzyme production.

Effect of pH and Temperature on Enzyme Activity and Stability
The appropriate enzymes for laundry detergent additives should have both high activity and high stability over a broad range of pH and temperatures. The pH activity profiles of the alkaline protease of B. gibsonii 6BS15-4 were determined with various pH ranges from 3 to 13. The result showed maximum activity at pH 12 and retention of around 80% of relative activity over a pH range of 10-12, while the enzyme activity was observed at around 50% of relative activity at pH 13. The results of pH stability revealed that this enzyme was 100% stable even at pH 12 for 60 min. It retained more than 85% of relative activity over a pH range of 6 to 12 when incubated at 45°C for 60 min (Fig. 3).
Temperature activity profiles of the alkaline protease of B. gibsonii 6BS15-4 were determined with temperature ranges of 30-80°C. The effect of temperatures on alkaline protease enzyme activity was observed and the results are shown in Fig. 4. Alkaline protease enzyme activity was highest in the range of 55-60°C, with an optimum temperature of 60°C for 60 min, but rapidly decreased at temperatures higher than 60°C, while enzyme activity was observed at around 50% of relative activity at 65°C. The alkaline protease of B. gibsonii 6BS15-4 was stable at 30-55°C for 60 min of incubation, pH 11. However, while the activity was rapidly lost above 55°C, around 50% of the relative activity was retained at 60°C.  The buffers were 0.05 M acetate buffer (pH 3-6), 0.05 M phosphate buffer (pH 6-8), 0.05 M glycine-NaOH buffer (pH 8-11), and 0.05 M hydroxide-chloride buffer (pH 11-13).
The alkaline protease of B. gibsonii 6BS15-4 activity was stable in the presence of 1% (v/v) Tween 80, 0.1% (v/v) hydrogen peroxide, and 1.5% (v/v) sodium perborate. In contrast, the enzyme activity decreased by 21.5% in the presence 0.1% (w/v) SDS. Thiol reagent and ethylenediaminetetraacetic acid did not inhibit enzyme activity, whereas phenylmethylsulfonyl fluoride did so significantly. Therefore, this enzyme could likely be a serine-type protease (Table 4).   Alkaline protease activity measured in the absence of any metal ions was reserved as control (100%). The enzyme activity was measured at pH 11 and 45°C.

Washing Performance Test with Alkaline Protease
The different treatment conditions of crude enzyme from B. gibsonii 6BS15-4 on blood stain removal are shown in Fig. 5. The alkaline protease of B. gibsonii 6BS15-4 plus detergent appeared to improve the cleansing process as evidenced by rapid blood stain removal from cotton cloth pieces when compared to enzyme or detergent alone.

Hydrolysis of Protein Substrates
The ability of alkaline protease to hydrolyze blood, egg solution, chicken feathers and milk was investigated at 30°C for 3 days. At day 1 of incubation, milk yielded the highest protein hydrolysis, chicken feathers the lowest. 99.94 ± 0.22 5 mM β-mercaptoethanol (BME) 102.85 ± 0.17 Alkaline protease activity measured in the absence of any chemical agent was reserved as control (100%). Enzyme activity was measured at pH 11 and 45°C. However, alkaline protease of B. gibsonii 6BS15-4 showed the highest capacity for hydrolysis of egg solution at 3 days of incubation (Fig. 6).

Discussion
The isolation and screening of new, alkaline protease-producing bacteria from an abundance of natural resources may reveal new opportunities to observe some novel alkaline protease-producing bacteria that could be used in highly alkaline conditions in various industries. The highest alkaline protease-producing bacterium isolated in this study was B. gibsonii 6BS15-4. Proteases produced by Bacillus species are the most important group of protease enzymes currently being industrially exploited. The results presented here agree with the literature, as several Bacillus species are known to be good alkaline protease producers and have been widely used in various industries [15][16][17][18][19].
The study on growth pattern and alkaline protease production of B. gibsonii 6BS15-4 was carried out in a shake flask containing 100 ml of BMSM at pH 11 and incubated at 30°C with shaking at 120 rpm for 192 h. The maximum proteolytic activity (316.88 units/ml) was observed after 168 h of growth where it reached stationary phase. B. gibsonii 6BS15-4 grew rapidly but scarcely produced alkaline protease in early log phase. However, in log phase (24-48 h), the protease increased substantially, and the maximum proteolytic activity (316.88 units/ml) was observed after 168 h of growth where it reached dead phase. The growth cycle of this bacteria has a very short stationary phase, possibly due to a stress environment condition or limited nutrients. The enzymes were found to be highly productive during the dead phase, possibly due to limited nutrients. According to the above results, it could be assumed that alkaline protease production was partially associated with microbial cell growth. Similar dynamics of protease production from B. circulans [29] and B. subtilis [30] have also been reported. The enzyme activity of B. firmus MTCC7728 was studied in relation to growth and the maximum reached at stationary phase was found to be 215 units/ml [31].
Food industries are notorious for high water consumption and production of enormous amounts of effluents, sludge, and waste. A dairy effluent supplement collected from cow dairy effluent was used in this study. Milk is comprised of nutritive compounds such as caseins and whey proteins. Caseins constitute 80% of the total protein present in milk [32]. Therefore, microorganisms are considered sources of cellular growth and protease production. Alkaline protease activity production from B. gibsonii 6BS15-4 using dairy effluent supplement with basal medium obtained 72.95% of alkaline protease activity when compared with alkaline protein cultivation from conventional media.
The dairy effluent supplement with basal medium with no added skim milk could save 38% of costs. However, enzyme productivity was 73%, thus the total saving was 15% when compared with the conventional media. The use of dairy effluent to produce alkaline protease is the eco-friendly and economical method for bioconversion of effluent wastes into value-added products, in addition, environmental pollution could be decreased. Moreover, the low cost of enzyme production eventually leads to a decrease in production cost.
Protease with the unique properties of pH stability and thermostability towards solvents, oxidizing agents, and surfactants are of particular use in industry. Alkaline proteases generally have optimal activities over a broad pH range of pH 5 to 11 and stability of protease enzyme in a broad pH range of pH 6 to 11 in B. gibsonii aprBG [28], B. licheniformis MH31 [33] and B. licheniformis Bl8 [34]. Meanwhile, in this study the enzyme showed maximum activity at pH 12 and retained activity over a pH range of 6 to 12, higher than in other reports. This confirmed the promising potential of alkaline protease from B. gibsonii 6BS15-4 for future industrial application, which requires enzyme stability at a wide pH range. Furthermore, the protease was active over a broad range of temperatures and had an optimum activity at 60°C, while other alkaline proteases of Bacillus spp. with optimal temperature at 50°C have also been reported [34]. The thermostability investigations of the alkaline protease from B. gibsonii 6BS15-4 indicated that the protease was absolutely stable below 55°C for 60 min incubation and pH 11. However, an optimum temperature of 50°C has been reported in B. gibsonii aprBG [28] and B. licheniformis Bl8 [34].
The enzyme showed maximum activity at pH 12 and retained activity over a pH range of 6 to 12 (Fig. 2). Meanwhile, B. gibsonii aprBG was also observed from protease over a range of pH from 5 to 11, with optimal activity at pH 9.5 [28].
None of the metal ions showed a considerable enhancing effect on the activity of the alkaline protease. However, K + , Cu 2+ and Mg 2+ have demonstrated enhancing effects on the activity of the reported alkaline proteases from some other bacterial sources, such as B. alveayuensis CAS 5 [18], B. subtilisSD11 [35], and B. safensis strain RH12 [36]. Here, the surfactants and oxidizing agents showed no effect on B. gibsonii 6BS15-4 alkaline protease activity. Thus, alkaline protease in this study was significantly more stable in the presence of non-ionic, anionic, and commercial detergents, which would make this enzyme a viable detergent additive. Additionally, the results of the blood stain removal experiment showed complete stain removal in detergent solution supplemented with enzyme, whereas the blood stain was not completely removed by the detergent solution alone.
Considering the production costs and reutilization of bioresources, alkaline protease production by microbial action seems to be promising approach; the use of dairy wastewater to produce protease is an inexpensive and ecofriendly method for the bioconversion of these wastes into value-added products and, for reducing environmental pollution. The alkaline protease obtained from B. gibsonii 6BS15-4 has high stability towards temperature and pH, and is also stable in the presence of non-ionic, anionic, and commercial detergents. These properties indicate its possible use in the commercial manufacture of detergent. Therefore, the alkaline protease in our study is proposed as an additive for detergents to enhance washing performance.